The present Ph.D. project constituted a first step in understanding mechanisms of reproductive isolation between two recently diverged sister species: the Lusitanian pine vole Microtus lusitanicus and the Mediterranean pine vole Microtus duodecimcostatus. Reproductive isolation is essential to speciation, and two types of isolation, preand post-mating, may prevent hybridization between two species. While premating barriers prevent copulation and promote conspecific reproduction, postmating barriers affect the success of heterospecific fertilization and hybrid viability, and potentiate its sterility. M. lusitanicus diverged from M. duodecimcostatus approximately 60,000 years ago, constituting one of the most recent speciation events among Microtus sp. voles. While M. lusitanicus inhabits the Northern region of the Iberian Peninsula, reaching the French Pyrenees, M. duodecimcostatus occupies Southern Iberia and part of the South of France. There is also a sympatry area of distribution, where both species occur, located in the centre of the Iberian Peninsula, covering parts of Portugal and Spain. Analyses on cytochrome b and microsatellites have uncovered a cytonuclear discordance over a large geographic area in Portugal, indicating a historical introgression of mitochondrial DNA from M. duodecimcostatus to M. lusitanicus. An incomplete reproductive isolation in nature is also suggested between both voles since two possible hybrids were detected in a sample size of nearly three hundred individuals. Moreover, behavioural isolation was hinted at, since there is a preference for conspecific over heterospecific odour cues. The gametic isolation barrier was proposed since heterospecific mating, in laboratory conditions, is less reproductively prolific than conspecific mating. This result suggests that fertilization between M. lusitanicus and M. duodecimcostatus may not always occur after copulation, probably due to incompatibilities in the sperm-oocyte heterospecific recognition. Considering these previous findings, the present Ph.D. project focused on M.lusitanicus and M. duodecimcostatus reproductive isolation, particularly on premating behavioural and post-mating gametic isolation barriers. It comprises five specific aims: 1) identify candidate genes related to odour cues communication; 2) analyse the expression of urinary proteins in both species; 3) infer if both species favour conspecific to heterospecific mating; 4) determine if both species present a pair bond, indicative of a monogamous mating system; and 5) evaluate the role of the sperm-binding protein zona pellucida 3, as a gametic isolation barrier. Four hypotheses were tested: 1) odour cues communication is an active behavioural reproductive barrier between M. lusitanicus and M. duodecimcostatus; 2) M. lusitanicus and M. duodecimcostatus prefer conspecific to heterospecific mating in the presence of potential mates of both species; 3) M. lusitanicus and M. duodecimcostatus are socially monogamous; and 4) the putative sperm-binding region of zona pellucida 3 is a gametic isolation barrier that impairs heterospecific mating between M. lusitanicus and M. duodecimcostatus. The results of the present Ph.D. project suggest that reproductive isolation between these sister species relies on multiple barriers and is still incomplete, enabling sporadic hybridization in nature. Overall, results also indicate that urinary proteins may play a role in species-specific discrimination; confirm social monogamy as the mating system of both voles, being a possible indirect behavioural isolation barrier at syntopy; reveal that individual behavioural variability may contribute to the behavioural isolation between M. lusitanicus and M. duodecimcostatus; and refute the putative sperm-binding region of ZP3 as a gametic barrier.